2.15 Immunofluorescence staining
For staining of HUVECs, cells cultured in 6-well plates were fixed with
4% paraformaldehyde for 30 min and permeabilized with 0.5% Triton-X
100 (Solarbio Science & Technology, China) for 15 min. Next, samples
were incubated with 5% BSA for 30 min at room temperature to block
nonspecific binding and then incubated with Ki67 (Cell Signaling
Technology; Cat#11882; 1:200) at 4 °C overnight. Alexa Fluor
488-conjugated anti-mouse IgG secondary antibody (Abcam; Cat#ab150113;
1:200) was then added and incubated for 1 h at room temperature. The
nuclei were labeled with DAPI for 10 min, and then images were captured
by Leica SP8 confocal microscopy (Leica, Wetzlar, Germany).
For quantification of the CD31 positive areas, sections were stained
with anti-CD31 antibody (Abcam; Cat#ab24590; 1:100) and then incubated
with Alexa Fluor 647-conjugated anti-rabbit IgG secondary antibody
(Abcam; Cat#ab150115, 1:200) for 60 min at room temperature.
Subsequently, cell nuclei were stained with DAPI. All fluorescent images
were taken using a Leica SP8 confocal microscopy (Leica, Wetzlar,
Germany).